Fig. 7: Rescue experiments in SK-MEL-5 TPC2 and Rab7a KO.

Proliferation of Rab7a KO (a) or TPC2 KO (b) cells expressing vector alone, Rab7^WT- or Rab7^Q67L-mCherry, assessed for 24-, 48-, and 72-h and normalized to vector. Proliferation assessed for 24-, 48-, and 72-h for Rab7a KO (c) or TPC2 KO (d) SK-MEL-5 cells expressing TPC2^WT-mCherry normalized to TPC2^L265P-mCherry, TPC2^M484L-YFP normalized to TPC2^WT-YFP, and treatment with the agonist TPC2-A1P normalized to DMSO control. e–h Representative images of the invasive phenotype determined by OE of mCherry vector, Rab7^WT- or Rab7^Q67L-mCherry in Rab7a KO and TPC2 KO, and statistical analysis shown in (e) and (g), respectively. Statistical significance was determined using one-way ANOVA, mean values ± SEM. i–l Representative images and statistical analysis of the invasive phenotype determined by OE of TPC2^WT-mCherry, TPC2^M484L-YFP, and/or treatment with TPC2-A1P in Rab7a and TPC2 KO, normalized to resp. control. Statistical significance in a–e, g, i, k was determined using one-way ANOVA followed by Bonferroni multiple comparisons test, mean values ± SEM (a–d: n = 9–15; e, g: n = 4; i: n = 3–9, k: n = 5–8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. m–p In vivo experiments showing tumor growth/weight after subcutaneous injection of B16F10luc WT, TPC2 KO or Rab7a KO cells into 5–6 week old C57Bl/6-Tyr mice. Treatment with vehicle control, ML-098 (0.04 mg/g) or TPC2-A1-P (0.02 mg/g) was performed daily (m, “Created in BioRender”). BioRender.com/n88t710.”). Bioluminescence images (d7 after injection) and bioluminescence signal intensities of tumors, mean ± SEM (n = 15 for Rab7a KO + TPC2-A1-P, n = 17 for Rab7a KO). Statistical significance was assessed by two-tailed unpaired t-test, **p < 0.0001 (mean ± SEM) (n). Tumor weights and representative tumors at the endpoint (day 14) are shown in (o, p). Statistical significance was determined using one-way ANOVA, mean values ± SEM (WT n = 16, TPC2 KO ± ML-098 n = 14 each, Rab7a KO n = 17, Rab7a KO + TPC2-A1-P n = 15), *p < 0.05, **p < 0.01. q In vivo experiments showing tumor cell dissemination after intravenous injection of 2 × 10⁵ B16F10-luc WT, TPC2 KO or Rab7a KO cells. Data from day 14 (endpoint) are shown. Statistical significance was determined using ordinary one-way ANOVA, mean ± SD (WT n = 7, TPC2 KO and Rab7a KO n = 9), *p < 0.05, **p < 0.01. Source data are provided as Source Data file.